Three-dimensional (3D) cell-based tissue grafts have been increasingly useful in tissue engineering and regenerative medicine. A critical building block in tissue engineering is the scaffold which can act as the supporting medium to deliver cell populations and induce the ingrowth of vessels and surrounding tissues. Therefore, it is necessary to develop tools to characterize the architecture of the scaffold. In addition, to study cell-scaffold interaction, namely, cell viability, migration, proliferation, and signaling within te scaffold, a non- destructive technique that can quantitatively image 3D cell behavior is required. The emerging multi-modality systems such as the combination of optical coherence tomography (OCT) with fluorescence confocal microscopy (FCM) and two-photon microscopy (TPM) that provide co-registered images of structural and functional properties of scaffolds and cells have had significant impact upon the field. However, both FCM and TPM have limited penetration depths thus precluding their characterization of cells deep inside the scaffolds. Therefore, there is a critical need for developing new methods that can analyze the engineered tissue structure in a non-destructive manner and with the ability to image deeper than microscopy. We propose a new platform of OCT and fluorescence laminar optical tomography (FLOT) for characterization cell-scaffold interaction. Two key findings from our lab provide the major motivation for this work: 1) We have demonstrated that OCT can image macroporous scaffolds and quantify their structural parameters including pore sizes, porosity, and interconnectivity; and the co-registered FCM can quantitatively describe cell composition on the scaffold surface. 2) We have developed a combined OCT and FLOT system for depth-resolved imaging of tissue morphology and molecular information in vivo up to 1-2 mm deep. The combined use of OCT and FLOT is a promising approach to characterize both structural and cellular information simultaneously to investigate cell-scaffold interaction, which enables longitudinal studies of cell viability, migraton, proliferation, and differentiation temporally as well as spatially within the scaffolds. We hypothesize that OCT and FLOT will enable 3D description of a scaffold's structure and cellular/molecular distribution, therefore elucidating cellular interactions within scaffolds. We propose to achieve our objective through three specific aims: 1) Establish the Capability of OCT/FLOT on Imaging Morphological and Functional Parameters in Engineered Tissues. 2) Image Stem-Cell-Laden Tissue Scaffolds and Correlate with Bone Formation In Vivo. 3) Prospectively Validate the Hypothesis that OCT/FLOT Imaging Can Predict Regenerative Outcomes. This project will result in a new non-destructive imaging technology for quantitative characterization of cell-scaffold interactions, which is essential to enable optimized design and materials of tissue engineering scaffolds, cell-seeding methods, and chemical/environmental cues. These studies will define a novel path towards advancing bone tissue engineering.